A mandated reduction in cigarette smoke of selected carcinogens and toxicants has been recommended by the World Health Organization Study Group on Tobacco Product Regulation (TobReg) and is now possible in the U.S. under the Family Smoking Prevention and Tobacco Control Act. Although a mandated reduction of individual toxicants and carcinogens may not necessarily lead to reduction in health risks, some potent carcinogens in cigarette smoke can be substantially reduced by modifying cigarette manufacturing approaches. Therefore, the overall goal would be to progressively reduce levels of these constituents in mainstream smoke as measured by standardized machine determined methods. However, the issue of how to test and regulate the contents of cigarette smoke represents a critical challenge. The currently used standard machine testing methods do not account for the complexities of smoker-cigarette interaction and are widely recognized to be inadequate for the prediction of human exposures. TobReg study group recommended that levels of toxicants be established per mg of nicotine. However, it is not known how the constituent per mg nicotine emissions in cigarette smoke are related to individual constituent exposures in smokers, and which factors may affect this relationship. Moreover, it is not clear which of the traditionally used standard smoking machine regimens may deliver constituent per mg nicotine levels in the U.S. cigarettes that are most closely related to the smokers' exposure. The goal of our proposal is to address these critical gaps. Building on our expertise in the analysis of tobacco products and biomarkers of exposure, we will focus on the carcinogenic tobacco-specific nitrosamines N2-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) to conduct research as described in the following specific aims: (1) To examine NNN and NNK per mg nicotine emissions in various U.S. cigarette brands under different smoking regimens. (2) To examine the extent to which changes in NNN and NNK per mg nicotine yields in smoke succeed in predicting changes in smokers' exposure to these carcinogens. (3) To determine which individual factors (for example, duration and intensity of smoking, nicotine metabolism, demographics) may affect the relationship between the machine- measured TSNA per mg nicotine and exposures in smokers. These factors may need to be considered when examining constituent/mg nicotine smoke yields for regulatory purposes. This research is critical for expanding the science base that informs the FDA as it develops, evaluates, and implements tobacco product regulations.